ACE2 is a membrane protein that regulates the cardiovascular system. Additionally, ACE2 acts as a receptor for host cell infection by human coronaviruses, including SARS-CoV-2 that emerged as the ...cause of the on-going COVID-19 pandemic and has brought unprecedented burden to economy and health. ACE2 binds the spike protein of SARS-CoV-2 with high affinity and shows little variation in amino acid sequence meaning natural resistance is rare. The discovery of a novel short ACE2 isoform (deltaACE2) provides evidence for inter-individual differences in SARS-CoV-2 susceptibility and severity, and likelihood of developing subsequent 'Long COVID'. Critically, deltaACE2 loses SARS-CoV-2 spike protein binding sites in the extracellular domain, and is predicted to confer reduced susceptibility to viral infection. We aimed to assess the differential expression of full-length ACE2 versus deltaACE2 in a panel of human tissues (kidney, heart, lung, and liver) that are implicated in COVID-19, and confirm ACE2 protein in these tissues. Using dual antibody staining, we show that deltaACE2 localises, and is enriched, in lung airway epithelia and bile duct epithelia in the liver. Finally, we also confirm that a fluorescently tagged SARS-CoV-2 spike protein monomer shows low binding at lung and bile duct epithelia where dACE2 is enriched.
The predicted protein encoded by the APJ gene discovered in 1993 was originally classified as a class A G protein-coupled orphan receptor but was subsequently paired with a novel peptide ligand, ...apelin-36 in 1998. Substantial research identified a family of shorter peptides activating the apelin receptor, including apelin-17, apelin-13, and Pyr
apelin-13, with the latter peptide predominating in human plasma and cardiovascular system. A range of pharmacological tools have been developed, including radiolabeled ligands, analogs with improved plasma stability, peptides, and small molecules including biased agonists and antagonists, leading to the recommendation that the APJ gene be renamed APLNR and encode the apelin receptor protein. Recently, a second endogenous ligand has been identified and called Elabela/Toddler, a 54-amino acid peptide originally identified in the genomes of fish and humans but misclassified as noncoding. This precursor is also able to be cleaved to shorter sequences (32, 21, and 11 amino acids), and all are able to activate the apelin receptor and are blocked by apelin receptor antagonists. This review summarizes the pharmacology of these ligands and the apelin receptor, highlights the emerging physiologic and pathophysiological roles in a number of diseases, and recommends that Elabela/Toddler is a second endogenous peptide ligand of the apelin receptor protein.
The apelin receptor, a G protein-coupled receptor, has emerged as a key regulator of cardiovascular development, physiology, and disease. However, there is a lack of suitable human in vitro models to ...investigate the apelinergic system in cardiovascular cell types. For the first time we have used human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and a novel inducible knockdown system to examine the role of the apelin receptor in both cardiomyocyte development and to determine the consequences of loss of apelin receptor function as a model of disease.
Expression of the apelin receptor and its ligands in hESCs and hESC-CMs was determined. hESCs carrying a tetracycline-inducible short hairpin RNA targeting the apelin receptor were generated using the sOPTiKD system. Phenotypic assays characterized the consequences of either apelin receptor knockdown before hESC-CM differentiation (early knockdown) or in 3D engineered heart tissues as a disease model (late knockdown). hESC-CMs expressed the apelin signalling system at a similar level to the adult heart. Early apelin receptor knockdown decreased cardiomyocyte differentiation efficiency and prolonged voltage sensing, associated with asynchronous contraction. Late apelin receptor knockdown had detrimental consequences on 3D engineered heart tissue contractile properties, decreasing contractility and increasing stiffness.
We have successfully knocked down the apelin receptor, using an inducible system, to demonstrate a key role in hESC-CM differentiation. Knockdown in 3D engineered heart tissues recapitulated the phenotype of apelin receptor down-regulation in a failing heart, providing a potential platform for modelling heart failure and testing novel therapeutic strategies.
The apelin receptor binds two distinct endogenous peptides, apelin and ELA, which act in an autocrine/paracrine manner to regulate the human cardiovascular system. As a class A GPCR, targeting the ...apelin receptor is an attractive therapeutic strategy. With improvements in imaging techniques, and the stability and brightness of dyes, fluorescent ligands are becoming increasingly useful in studying protein targets. Here, we describe the design and validation of four novel fluorescent ligands; two based on Pyr1apelin-13 (apelin488 and apelin647), and two based on ELA-14 (ELA488 and ELA647).
Fluorescent ligands were pharmacologically assessed using radioligand and functional in vitro assays. Apelin647 was validated in high content imaging and internalisation studies, and in a clinically relevant human embryonic stem cell-derived cardiomyocyte model. Apelin488 and ELA488 were used to visualise apelin receptor binding in human renal tissue.
All four fluorescent ligands retained the ability to bind and activate the apelin receptor and, crucially, triggered receptor internalisation. In high content imaging studies, apelin647 bound specifically to CHO-K1 cells stably expressing apelin receptor, providing proof-of-principle for a platform that could screen novel hits targeting this GPCR. The ligand also bound specifically to endogenous apelin receptor in stem cell-derived cardiomyocytes. Apelin488 and ELA488 bound specifically to apelin receptor, localising to blood vessels and tubules of the renal cortex.
Our data indicate that the described novel fluorescent ligands expand the pharmacological toolbox for studying the apelin receptor across multiple platforms to facilitate drug discovery.
Patients with cardiovascular comorbidities are more susceptible to severe infection with SARS-CoV-2, known to directly cause pathological damage to cardiovascular tissue. We outline a screening ...platform using human embryonic stem cell-derived cardiomyocytes, confirmed to express the protein machinery critical for SARS-CoV-2 infection, and a SARS-CoV-2 spike-pseudotyped virus system. The method has allowed us to identify benztropine and DX600 as novel inhibitors of SARS-CoV-2 infection in a clinically relevant stem cell-derived cardiomyocyte line. Discovery of new medicines will be critical for protecting the heart in patients with SARS-CoV-2, and for individuals where vaccination is contraindicated.
Marfan syndrome (MFS) is a rare connective tissue disorder caused by mutations in FBN1. Patients with MFS notably suffer from aortic aneurysm and dissection. Despite considerable effort, animal ...models have proven to be poorly predictive for therapeutic intervention in human aortic disease. Patient-derived induced pluripotent stem cells can be differentiated into vascular smooth muscle cells (VSMCs) and recapitulate major features of MFS. We have screened 1,022 small molecules in our in vitro model, exploiting the highly proteolytic nature of MFS VSMCs, and identified 36 effective compounds. Further analysis identified GSK3β as a recurring target in the compound screen. GSK3β inhibition/knockdown did not ameliorate the proliferation defect in MFS-VSMCs but improved MFS-VSMC proteolysis and apoptosis and partially rescued fibrillin-1 deposition. To conclude, we have identified GSK3β as a novel target for MFS, forming the foundation for future work in MFS and other aortic diseases.
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•Developed an iPSC-based drug screen for MFS and tested 1,022 small molecules•Identified GSK3β as a recurring target among effective small molecules•Validated the outcome of the drug screen using six GSK3β inhibitors and siRNA•GSK3β inhibition/knockdown decreased proteolysis and apoptosis in 4 patient lines
Animal models of MFS have not yet been successful in predicting human disease. Sinha and colleagues have developed an iPSC model of MFS and used it as a platform for drug screening. They screened over a thousand compounds and identified GSK3β as a top drug target. This work is the foundation for further screening in MFS and related diseases.
Abstract only Regenerative cardiovascular medicine is emerging as a promising therapeutic alternative to heart transplantation in patients suffering from chronic heart failure. Although promising, ...challenges to cell therapy for cardiac repair remain, including cell survival, maturation, graft size, revascularisation, and immunogenicity. It has recently been shown that co-transplantation of human embryonic stem cell (hESC)-derived epicardial cells (EPI) with hESC-derived cardiomyocytes (CM) improves cardiac repair processes with respect to engraftment, cell maturation, and graft and host vascularisation. One plausible explanation for the observed benefits of co-transplantation is paracrine-mediated effects. While RNA sequencing data has been used to identify putative players in hESC-EPIs, little is known about their involvement in mediating hESC-CM maturation and revascularization, in particular in communication with endothelial cells (EC). Here, the role of paracrine signalling in hESC-EPI-mediated promotion of hESC-CM maturation and angiogenesis is being investigated with an emphasis on the involvement of extracellular vesicles (EVs). hESC-EPI-EVs improved responsiveness to pacing and coordination of contraction in vitro in hESC-CM cell culture and engineered heart tissues (EHTs). Moreover, hESC-EPI-EVs and hESC-EPI supernatant excluding EVs independently promoted tube formation in in vitro gel-based angiogenesis assays. hESC-EPI supernatant excluding EVs also promoted EC proliferation in MTS assays and EC migration in wound closure assays. Characterisation of the hESC-EPI-EV-cargo on a protein level revealed candidate factors involved in cardiac repair processes. Together with hESC-EPI-EV miRNA-sequencing and hESC-EPI gene expression data key paracrine signalling pathways and candidate factors to manipulate them are being identified and investigated in in vitro angiogenesis and CM maturation assays. Promising angiogenesis promoting candidates are being validated in vivo using yolk-sac membrane and chorioallantoic membrane assays. Identification of the paracrine signalling pathways involved would make it possible to effectively address selected cardiac repair processes, resulting in new approaches to therapy.
Abstract only Introduction: Cardiovascular comorbidities are a major risk factor in patients infected with SARS-CoV-2. SARS-CoV-2 Spike protein binds to host cell surface ACE2 to gain entry. ACE2 is ...subsequently downregulated by internalisation. We hypothesise that an ACE2 knockdown system in beating human embryonic stem cell-derived cardiomyocytes (hESC-CMs) will recapitulate the downregulation of ACE2, and reduce the ability of a SARS-CoV-2 Spike protein pseudotyped virus to infect these clinically relevant cells. Method: ACE2 was knocked down (KD) with CRISPR/Cas9 in hESCs, before differentiating to hESC-CMs and sub-culturing in 96 well plates. Sanger sequencing confirmed amino acid changes. Catalytic ACE2 activity was measured by mass spectrometry in wild-type versus KD by conversion of apelin-13 to apelin-12 in hESC-CMs supernatant. ACE2 activity was also measured by fluorescent substrate assay. Pseudotyped virus infection was visualised by high content screening in ACE2 KD versus wild-type (n=4). All data are mean±SEM. Results: Sequencing revealed two substitutions at ACE2 222-223 , and three deletions at ACE2 224-226 . This reduced ACE2 catalytic activity by ~60-70% by apelin-12 accumulation using mass spectrometry (Fig 1a), and fluorescent assay. Furthermore, KD reduced infection of hESC-CMs by pseudotyped virus to 27.3±9.6% of the cell population versus 74.8±4.7% for wild-type (Fig 1b,c). This is consistent with our previous data, showing DX600 (peptidic inhibitor of ACE2 catalytic site) significantly reduced infection of wild-type hESC-CMs to 20.5±6.5%. Conclusions: In conclusion, we generated a functional ACE2 knockdown in a beating cardiomyocyte cell model. Both the catalytic activity and the ability to bind SARS-CoV-2 Spike protein of ACE2 reduced, importantly indicating ACE2 is rate limiting for infection. We aim to use this system to further explore the cardiovascular consequences of SARS-CoV-2 infection and subsequent downregulation of ACE2.
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